Phage-inspired strategies to combat antibacterial resistance.

Antimicrobial resistance (AMR) in clinically priority pathogensis now a major threat to public health worldwide. Phages are bacterial parasites that efficiently infect or kill specific strains and represent the most abundant biological entities on earth, showing great attraction as potential antibacterial therapeutics in combating AMR. This review provides a summary of phage-inspired strategies to combat AMR. We firstly cover the phage diversity, and then explain the biological principles of phage therapy that support the use of phages in the post-antimicrobial era. Furthermore, we state the versatility methods of phage therapy both from direct access as well as collateral access. Among the direct access approaches, we discuss the use of phage cocktail therapy, phage-encoded endolysins and the bioengineering for function improvement of used phages or endolysins. On the other hand, we introduce the collateral access, including the phages antimicrobial immunity combined therapy and phage-based novel antibacterial mimic molecules. Nowadays, more and more talented and enthusiastic scientist, doctors, pharmacists, media, authorities, and industry are promoting the progress of phage therapy, and proposed more phages-inspired strategy to make them more tractable to combat AMR and benefit more people, more animal and diverse environment in "one health" framework.

Coevolutionary phage training and Joint application delays the emergence of phage resistance in Pseudomonas aeruginosa.

Antibiotic-resistant bacteria are current threats to available antibiotic therapies, and this has renewed interest in the therapeutic use of phage as an alternative. However, development of phage resistance has led to unsuccessful therapeutic outcomes. In the current study, we applied phage training to minimize bacterial phage resistance and to improve treatment outcome by adapting the phage to their target hosts during co-evolution. We isolated and characterized a novel Pseudomonas aeruginosa N4-like lytic phage (PWJ) from wastewater in Yangzhou, China. PWJ is a double-stranded DNA podovirus that can efficiently lyse the model strain ATCC 27,853 and opportunistic pathogen PAO1. Genome sequencing of PWJ revealed features similar to those of the N4-like P. aeruginosa phage YH6. We used PWJ to screen for an evolved trained phage (WJ_Ev14) that restored infectivity to PWJ phage bacterial resisters. BLASTN analysis revealed that WJ_Ev14 is identical to its ancestor PWJ except for the amino acid substitution R1051S in its tail fiber protein. Moreover, phage adsorption tests and transmission electron microscopy of resistant bacteria demonstrated that the R1051S substitution was most likely the reason WJ_Ev14 could re-adsorb and regain infectivity. Furthermore, phage therapy assays in vitro and in a mouse P. aeruginosa lung infection model demonstrated that PWJ treatment resulted in improved clinical results and a reduction in lung bacterial load whereas the joint phage cocktail (PWJ+ WJ_Ev14) was better able to delay the emergence of resister bacteria. The phage cocktail (PWJ +WJ_Ev14) represents a promising candidate for inclusion in phage cocktails developed for clinical applications.

Hollow Porous CoO@Reduced Graphene Oxide Self-Supporting Flexible Membrane for High Performance Lithium-Ion Storage.

We report an environment-friendly preparation method of rGO-based flexible self-supporting membrane electrodes, combining Co-MOF with graphene oxide and quickly preparing a hollow CoO@rGO flexible self-supporting membrane composite with a porous structure. This unique hollow porous structure can shorten the ion transport path and provide more active sites for lithium ions. The high conductivity of reduced graphene oxide further facilitates the rapid charge transfer and provides sufficient buffer space for the hollow Co-MOF nanocubes during the charging process. We evaluated its electrochemical performance in a coin cell, which showed good rate capability and cycling stability. The CoO@rGO flexible electrode maintains a high specific capacity of 1103 mAh g-1 after 600 cycles at 1.0 A g-1. The high capacity of prepared material is attributed to the synergistic effect of the hollow porous structure and the 3D reduced graphene oxide network. This would be considered a promising new strategy for synthesizing hollow porous-structured rGO-based self-supported flexible electrodes.

Similarities of P1-Like Phage Plasmids and Their Role in the Dissemination of blaCTX-M-55.

The P1-like phage plasmid (PP) has been widely used as a molecular biology tool, but its role as an active accessory cargo element is not fully understood. In this study, we provide insights into the structural features and gene content similarities of 77 P1-like PPs in the RefSeq database. We also describe a P1-like PP carrying a blaCTX-M-55 gene, JL22, which was isolated from a clinical strain of Escherichia coli from a duck farm. P1-like PPs were very similar and conserved based on gene content similarities, with only eight highly variable regions. Importantly, two kinds of replicon types, namely, IncY and p0111, were identified and can be used to specifically identify the P1-like phage. JL22 is similar to P1, acquiring an important foreign DNA fragment with two obvious features, namely, the plasmid replication gene repA' (p0111) replacing the gene repA (IncY) and a 4,200-bp fragment mobilized by IS1380 and IS5 and containing a blaCTX-M-55 gene and a trpB gene encoding tryptophan synthase (indole salvaging). The JL22 phage could be induced but had no lytic capacities. However, a lysogenic recipient and intact structure of JL22 virions were observed, showing that the extended-spectrum ß-lactamase blaCTX-M-55 gene was successfully transferred. Overall, conserved genes can be a good complement to improve the identification efficiency and accuracy in future screening for P1-like PPs. Moreover, the highly conserved structures may be important for their prevalence and dissemination. IMPORTANCE As a PP, P1 DNA exists as a low-copy-number plasmid and replicates autonomously with a lysogenization style. This unique mode of P1-like elements probably indicates a stable contribution to antibiotic resistance. After analyzing these elements, we show that P1-like PPs are very similar and conserved, with only eight highly variable regions. Moreover, we observed the occurrence of replicon IncY and p0111 only in the P1-like PP community, implying that these conserved regions, coupled with IncY and p0111, can be an important complement in future screening of P1-like PPs. Identification and characterization of JL22 confirmed our findings that major changes were located in variable regions, including the first detection of blaCTX-M-55 in such a mobile genetic element. This suggests that these variable regions may facilitate foreign DNA mobilization. This study features a comprehensive genetic analysis of P1-like PPs, providing new insights into the dissemination mechanisms of antibiotic resistance through P1 PPs.

Effects of the Newly Isolated T4-like Phage on Transmission of Plasmid-Borne Antibiotic Resistance Genes via Generalized Transduction.

Bacteriophages are the most abundant biological entities on earth and may play an important role in the transmission of antibiotic resistance genes (ARG) from host bacteria. Although the specialized transduction mediated by the temperate phage targeting a specific insertion site is widely explored, the carrying characteristics of "transducing particles" for different ARG subtypes in the process of generalized transduction remains largely unclear. Here, we isolated a new T4-like lytic phage targeting transconjugant Escherichia coli C600 that contained plasmid pHNAH67 (KX246266) and encoded 11 different ARG subtypes. We found that phage AH67C600_Q9 can misload plasmid-borne ARGs and package host DNA randomly. Moreover, for any specific ARG subtype, the carrying frequency was negatively correlated with the multiplicity of infection (MOI). Further, whole genome sequencing (WGS) identified that only 0.338% (4/1183) of the contigs of an entire purified phage population contained ARG sequences; these were floR, sul2, aph(4)-Ia, and fosA. The low coverage indicated that long-read sequencing methods are needed to explore the mechanism of ARG transmission during generalized transduction.

Evaluation of Potential ARG Packaging by Two Environmental T7-Like Phage during Phage-Host Interaction.

The increase in antimicrobial resistance is a threat to both human and animal health. The transfer of antibiotic resistance genes (ARG) via plasmids has been studied in detail whereas the contribution of bacteriophage-mediated ARG transmission is relatively little explored. We isolated and characterized two T7-like lytic bacteriophages that infected multidrug-resistant Escherichia coli hosts. The morphology and genomic analysis indicated that both phage HZP2 and HZ2R8 were evolutionarily related and their genomes did not encode ARGs. However, ARG-like raw reads were detected in offspring sequencing data with a different abundance level implying that potential ARG packaging had occurred. PCR results demonstrated that six fragments of genes (qnrS, cmlA, tetM, blaTEM, sul3, mcr-1) were potentially packaged by phage HZP2 and four (qnrS, cmlA, blaTEM, mcr-1) by phage HZ2R8. Further quantitative results showed that ARG abundance hierarchies were similar. The gene blaTEM was the most abundant (up to 1.38 × 107 copies/mL) whereas cmlA and qnrS were the least. Moreover, the clinically important mcr-1 gene was the second most abundant ARG indicating a possibility for spread through generalized transduction. Together, our results indicated that these structurally similar phage possessed similar characteristics and potential packaging during phage-host interaction displayed an ARG preference rather than occurring randomly.

[Production of L-2-aminobutyric acid from L-threonine using a trienzyme cascade].

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.

Analysis of energy monitoring for a double-pulsed CO2 integrated path differential absorption lidar at 1.57 µm.

For double-pulsed 1.57 µm integrated path differential absorption lidar, the transmitted pulse energy measurement is an important factor that can influence the uncertainty of CO2 concentration measurement. An energy monitoring experiment was performed to determine how to improve the measurement precision of the transmitted pulse energy. Ground glass diffusers were used to reduce the speckle effect during energy monitoring. The roughness and rotational speed of the ground glass diffusers were considered and compared. The normalized energy ratios between on-line and off-line echo pulses and on-line and off-line energy monitoring pulses were analyzed, and the Allan deviation was used to evaluate the energy monitoring results. Averaging 148 shots, the standard deviation of the normalized energy ratio reached 0.0757%, whereas the correlation between the energy ratio of the on-line and off-line energy monitoring pulses and the energy ratio of the on-line and off-line echo pulses was higher than 90%.

Double-pulse 1.57 µm integrated path differential absorption lidar ground validation for atmospheric carbon dioxide measurement.

A ground-based double-pulse integrated path differential absorption (IPDA) instrument for carbon dioxide (CO2) concentration measurements at 1572 nm has been developed. A ground experiment was implemented under different conditions with a known wall located about 1.17 km away acting as the scattering hard target. Off-/offline testing of a laser transmitter was conducted to estimate the instrument systematic and random errors. Results showed a differential absorption optical depth (DAOD) offset of 0.0046 existing in the instrument. On-/offline testing was done to achieve the actual DAOD resulting from the CO2 absorption. With 18 s pulses average, it demonstrated that a CO2 concentration measurement of 432.71±2.42 ppm with 0.56% uncertainty was achieved. The IPDA ranging led to a measurement uncertainty of 1.5 m.

Injection seeded single-frequency pulsed Nd:YAG laser resonated by an intracavity phase modulator.

A reliable single frequency Q-switched Nd:YAG laser is developed by using a lithium niobate crystal as the intracavity phase modulator. Successful injection seeding is performed by adopting an electro-optic crystal in an effectively simplified cavity arrangement. The laser is capable of producing 4.8 mJ pulse-energy at 400 Hz repetition rate with nearly Fourier-transform-limited spectral linewidth. The pulse duration is approximately 25 ns, and the beam quality factor M2 is less than 1.3.

Deficiency of P-selectin or P-selectin glycoprotein ligand-1 leads to accelerated development of glomerulonephritis and increased expression of CC chemokine ligand 2 in lupus-prone mice.

The selectins and their ligands mediate leukocyte rolling on endothelial cells, the initial step in the emigration cascade leading to leukocyte infiltration of tissue. These adhesion molecules have been shown to be key promoters of acute leukocyte emigration events; however, their roles in the development of long-term inflammatory responses, including those that occur during chronic inflammatory diseases such as systemic lupus erythematosus, are unclear. To assess participation of P-selectin in such disorders, we studied the progression of systemic lupus erythematosus-like disease in P-selectin-deficient and control MRL/MpJ-Fas(lpr) (Fas(lpr)) mice. Surprisingly, we found that P-selectin deficiency resulted in significantly earlier mortality, characterized by a more rapid development of glomerulonephritis and dermatitis. Expression of CCL2 (MCP-1) was increased in the kidneys of P-selectin mutant mice and in supernatants of LPS-stimulated primary renal endothelial cell cultures from these mice. A closely similar phenotype, including elevated renal expression of CCL2, was also observed in Fas(lpr) mice deficient in the major P-selectin ligand, P-selectin glycoprotein ligand-1. These results indicate that P-selectin and P-selectin glycoprotein ligand-1 are not required for leukocyte infiltration and the development of autoimmune disease in Fas(lpr) mice, but rather expression of these adhesion molecules is important for modulating the progression of glomerulonephritis, possibly through down-regulation of endothelial CCL2 expression.

Loss of LFA-1, but not Mac-1, protects MRL/MpJ-Fas(lpr) mice from autoimmune disease.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by immune complex-mediated tissue injury. Many different adhesion molecules are thought to participate in the development of SLE; however, few studies have directly examined the contributions of these proteins. Here we demonstrate that LFA-1 plays an essential role in the development of lupus in MRL/MpJ-Fas(lpr) mice. Mice deficient in LFA-1, but not Mac-1, showed significantly increased survival, decreased anti-DNA autoantibody formation, and reduced glomerulonephritis. The phenotype of the LFA-1-deficient mice was similar to that observed in beta(2) integrin-deficient (CD18-null) MRL/MpJ-Fas(lpr) mice, suggesting a lack of redundancy among the beta(2) integrin family members and other adhesion molecules. These studies identify LFA-1 as a key contributor in the pathogenesis of autoimmune disease in this model, and further suggest that therapeutic strategies targeting this adhesion molecule may be beneficial for the treatment of SLE.